a) Field of the Invention
The invention is directed to so-called laser-compatible NIR marker dyes based on polymethines for use in optical, in particular, fluorescence optical determination and detection methods. Typical applications of the process are based on the reaction of dye marked antigens, antibodies, ligands or DNA segments with the respective complementary species.
Possible uses exist, for example, in the fields of medicine, pharmaceutics and in the areas of life science, materials science, in environmental monitoring and in the detection of organic and inorganic micro-samples occurring naturally and in technological contexts, but they are not limited to the aforementioned fields.
b) Description of the Related Art
The usability of polymethines as NIR markers has been known of for a long time; they distinguish themselves by their strong absorption maxima which can easily be transposed into the NIR range (Fabian, J.; Nakazumi, H.; Matsuoka, M.: Chem. Rev. 1992, 92, 1197). With a suitable substituent pattern and pi-electron system and at a sufficient quantum yield they also fluoresce in the red and near infrared (NIR) range. Correspondingly, these compounds are widely used in different technological fields: as sensitizers in AgX materials, as laser dyes, as quantum counters, as indicator dyes in sensor technology, as light absorbers in writable CDs and last but not least as biomarkers (xe2x80x9cNear-Infrared Dyes for High Technology Applicationxe2x80x9d, published by Daehne, S.; Resch-Genger, U.; Wolfbeis, O.-S., Kluwer, Academic Publishersxe2x80x94Dordrecht/Boston/Londonxe2x80x941998).
The number of polymethines used as biomarkers is limited. So far, only the trimethine Cy3 derived from astraphloxine (DE 410 487), the vinylogous pentamethine Cy5 and the doubly vinylogous heptamethine Cy7 with absorption maxima at approximately 550 nm, approximately 650 nm and approximately 750 nm have so far found wide commercial application in this manner (U.S. Pat. No. 5,627,027). Also available are the polysulfonated trimethine Cy3.5 derived from the commercial hepatmethine xe2x80x9cIndocynaninegreenxe2x80x9d or xe2x80x9cCardio Greenxe2x80x9d and the pentamethine Cy5.5 (U.S. Pat. No. 5,569,766). Heptamethines with aliphatic bridges in the polymethine chain have been developed by Patonay (U.S. Pat. No. 5,800,995). All commercial biomarkers are characterized by terminal heteroaromatics derived from indene or heteroindene (Fischer""s base). If methylsubstituted cycloimmonium salts are used as terminal polymethine building blocks, it is necessary to arrange at least five sequential sp2 hybridized carbon atoms (pentamethines) between the heterocycles to generate absorption maxima at the boundary to the NIR range.
The NIR polymethines used in technology as biomarkers have the distinct disadvantage that lengthening the polymethine chain increases the opportunities for nucleophilic or electrophilic attack on the chain, in consequence of which the pi-system is destroyed. Further disadvantages of these marker dyes consist in their insufficient photostability and stability in storage, complicated synthesis and purification stages, low absorption coefficients/low fluorescent quantum yields as well as undesired changes of their optical properties in the presence of or after bonding with proteins or nucleic acid oligomeres. For example, a reduction of the fluorescent quantum yield of Cy5 has been described for the covalent bonding with different albumins (Oswald, B.; Patsenker, L.; Duschl, J.; Szmacinski, H.; Wolfbeis, O. S.; Terpeschnig, E.; Bioconjugate Chem. 1999, 10, 925-931).
The use of pyrylium and benzopyrylium heterocycles or the corresponding mesomeric chromenes as terminal end groups in marker dyes in biologically relevant systems is so far not known in the art. This is due to the extreme sensitivity to hydrolysis of these pi-deficient aromatics, especially in an aqueous basic environment (H. Lietz, G. Haucke, P. Czerney, B. John, J. Prakt. Chem., 1996, 338, 725-730).
Telfer et al. (U.S. Pat. No. 5,262,549) describe symmetrical trimethines based on 2-alkyl substituted benzopyrylium salts for the use as NIR absorbers in polimeric media with a reduced tendency towards aggregation in these media.
The primary object of the invention is to create NIR marker dyes based on polymethine which have a high degree of photostability and stability in storage as well as a high fluorescent yield and which can be excited to fluorescence in the easiest possible manner by means of laser radiation in the longwave visible or NIR spectral range, particularly with light of a helium/neon or diode laser.
The present invention describes marker dyes based on non-symmetrical polymethines which contain a substituted xcfx89-(benz[b]pyran-4-ylidene)alk-1-enyl) unit of general formula (I), 
where Z is a substituted derivative of benzooxazol, benzothiazol, 2,3,3-trimethylindolenine, 2,3,3-trimethyl-4,5-benzo3H-indolenine, 3- and 4-picoline, lepidine, chinaldine and 9-methylacridine derivatives with the general formulae IIa or IIb or IIc 
and where
X stands for an element of the group O, S, Se or the structural element N-alkyl or C(alkyl)2,
N stands for the numerical value 1, 2 or 3,
R1-R14 are equal or different and can be hydrogen, one or more alkyl, aryl, heteroaryl or heterocycloalipathic fragments, a hydroxy or alkoxy group, an alkylsubstituted or cyclical amine function and/or two fragments in ortho position to each other, for example R10 and R11, can together form another aromatic ring,
At least one of the substituents R1-R14 can be a solubilizing or ionizable or ionized substituent, like cyclodextrine, sugar, SO3xe2x88x92, PO32xe2x88x92, COOxe2x88x92, or NR3+, which determines the hydrophilic properties of these dyes; here it is possible that this substituent can be bound to the marker dye by means of a spacer group,
At least one of the substituents R1-R14 can stand for a reactive group which facilitates a covalent linking of the dye to the aforementioned carrier molecules, while this substituent can also be bound to the dye by means of a spacer group, and
R1 is a substituent which has a quarternary C-atom in alpha-position relative to the pyran ring. Examples for such substituents are t-butyl (xe2x80x94C(CH3)3) and adamantyl (xe2x80x94C10H15/tricyclo[3.3.1.13,7]decyl).
Subclaims 2 to 20 list specific embodiment forms and applications of the marker dyes.
These substituted derivatives of indol, heteroindol, pyridine, chinoline or acridine of the general formula I can be used as dyes for the optical marking of organic or inorganic microparticles, for example of proteins, nucleic acids, DNA, sugars, biological cells, lipids, drugs or organic or inorganic polymeric carrier substances.
Here, the marking of particles can be done by the formation of ionic interaction between the markers of general formula I and the substances to be marked.
The functional groups of these markers activated with regards to nucleophiles can couple covalently with an OH, NH2 or SH function, which therefore creates a system for the qualitative and quantitative determination of organic and inorganic substances, like said proteins, nucleic acids, DNA, sugars, biological cells, lipids, drugs or organic or inorganic polymers.
The coupling reaction can take place in an aqueous or mostly aqueous solution, preferably at room temperature. During this a conjugate with fluorescent properties is created.
By means of the preparation of non-symmetrical polymethines, which on the one hand have an easily derivatizable heterocycle of the type of the pyridine, chinoline, indol, heteroindol or acridine derivatives and on the other hand have a novel 6-ring heterocycle, in particular the following advantages are achieved:
Trimethines already absorb in the spectral range  greater than 650 nm and have a significantly improved photochemical and thermal stability when compared with polymethines known so far in the art which have absorption maxima  greater than 650 nm (penta- and heptamethines).
By means of molecular engineering, it is possible to control the position and intensity of the absorption and emission maxima at will and to adapt them to emission wavelengths of different excitation lasers, in particular NIR laser diodes.
The marker dyes can be produced by a relatively simple two-stage synthesis with which a variety of dyes with functionalities that differ, for example, with regards to the total charge of the dye and the number, specificity and reactivity of the activated group used for the immobilization can be provided in a manner that is specific to the respective application.
Compounds with the general formula I as well as systems derived from them (conjugates) can be used in optical, in particular fluorescence optical qualitative and quantitative determination methods for the diagnosis of cell properties, in biosensors (point-of-care measurements), exploration of the genome and in miniaturization technology. Typical applications lie in the fields of cytometry, cell sorting, fluorescence correlation spectroscopy (FCS), ultra-high throughput screening (UHTS), multicolor fluorescence in situ hybridization (FISH) and in microarrays (gene and protein chips).
Here, a microarray is a grid-like arrangement of molecules immobilized on at least one surface which can be used for the study of the interaction between receptors and ligands. A grid-like arrangement means more than two molecules which are different from each other and which are immobilized in different, predefined regions of known positions on a surface.
A receptor is a molecule which has an affinity to a given ligand. Receptors can be naturally occurring or artificially produced molecules.
Receptors can be used in their pure state or bound to other species. Receptors can be bound covalently or non-covalently either directly or via certain coupling mediators to a bonding partner.
Examples of receptors which can be detected by means of this invention include agonists and antagonists for cell membrane receptors, toxins and other poisonous substances, viral epitopes, hormones like opiates and steroids, hormone receptors, peptides, enzymes, enzyme substrates, active substances that act as co-factors, lectines, sugars, oligonucleotides, nucleic acids, oligosaccharides, cells, cell fragments, tissue fragments, proteins and antibodies, but they are not limited to the named substances.
A ligand is a molecule that is recognized by a particular receptor. Examples of ligands which can be detected by this invention include agonists and antagonists for cell membrane receptors, toxins and other poisonous substances, viral epitopes, hormones like opiates and steroids, hormone receptors, peptides, enzymes, enzyme substrates, active substances that act as co-factors, lectines, sugars, oligonucleotides, nucleic acids, oligosaccharides, cells, cell fragments, tissue fragments, proteins and antibodies, but they are not limited to the named substances.
The invention is subsequently to be illustrated in more detail by means of embodiment examples and the drawings.